Field of the Invention
The invention relates to the selection of the most favorable ion species from a mixture of biopolymers for the acquisition of fragment ion mass spectra when the ionization creates biopolymer ions in different charge states.
Definitions
Instead of the statutory “unified atomic mass unit” (u), this document uses the unit “dalton” (Da), which was added in the last (eighth) 2006 edition of the document “The International System of Units (SI)” of the “Bureau International des Poids et Mesures” on an equal footing with the unified atomic mass unit. As is noted in the document, this was done primarily in order to allow the use of the units Kilodalton (kDa), millidalton (mDa) and similar compositions.
The term “ion species” is used here to mean all ions of a substance S in a defined charge state z, i.e. Sz+, where z is the number of elementary charges of the ion. An ion species includes all ions of different isotopic compositions. An ion species can be characterized by stating a value for M/z where M is not, however, the monoisotopic mass (usually designated as m), as is often the convention in mass spectrometry, but the molecular mass averaged over the isotopic compositions M (previously called the molecular weight).
Description of the Related Art
The identification of biopolymers, especially proteins, with molecular masses M between 5 and 100 kilodaltons in body fluids, is of great interest in pharmacology, biology and medicine. The following discussion relates mainly to proteins. Examples of very interesting proteins are antibodies, usually enzymatically split into three partial molecules with masses of around 50, 50 and 25 kilodaltons. The identification is preferably carried out by mass spectrometric analysis of fragment ion spectra after liquid chromatographic separation, although it is often not possible to completely separate many proteins chromatographically. The ionization is usually carried out by electrospraying (ESI). For each protein, the mass spectrum contains regular patterns of multiply charged ions with a broad, usually relatively smooth distribution of the intensities for the ions with different charge numbers z, the most intense ion species usually being located at mass-to-charge ratios M/z between 600 and 1200 daltons. Each ion species characterized by its mass-to-charge ratio M/z exhibits a narrow distribution of ions of different isotopic composition. When large numbers of different substances are present, there are many overlaps of the isotopic distributions.
If a protein is subjected to electrospray ionization, then the number of ion species of different charge states z depends on the mass M of the protein; proteins with high masses generally have a larger number of different charge states than those with low masses. In the electrospray method, a small protein such as ubiquitin, with an average molecular mass M=8564.76 Da, typically produces eight different charge states with charge numbers z=7, 8, 9, . . . , 14, when it is sprayed with a typical solvent mixture of water, acetonitrile and formic acid (FIG. 1). Bovine serum albumin with an average mass of M=66.4 kDa is present in the mass spectrum in 32 different charge states z (FIG. 2). The structure of the protein, the use of denaturizing solvents, and the existence of disulfide bonds within the protein can affect the charge distribution; the strong interlinking within the bovine serum albumin means that, during the ionization, relatively few protons can be taken up as charge carriers, which in turn means that the most intense ion species are relatively heavy and appear at around M/z=1200 Da.
The identification of a protein requires the acquisition of a mass spectrum of fragment ions of a selected ion species of this protein with a mass-to-charge ratio M/z, where usually all the isotopic signals of this ion species are included in the fragmentation. The mass spectrometric analysis is usually carried out in time-of-flight mass spectrometers with orthogonal ion injection (OTOF), wherein the isolation of the selected ion species and its fragmentation are usually carried out in quadrupole mass filters and ion storage devices. The multiply charged protein molecules are usually fragmented by transferring electrons from suitable, negatively charged donor molecule ions (ETD=electron transfer dissociation). The acquisition of a good mass spectrum of the fragment ions takes around five to ten seconds, and the proteins elute in the chromatogram within a window of only around 30 to 45 seconds, and can thus be mass spectrometrically evaluated only during this time. It is therefore important to be able to quickly and automatically select from an unfragmented mass spectrum the correct ion species to be fragmented for the individual proteins, and they must not overlap with other protein ions.
As can be seen in FIG. 3, with an ESI mass spectrum of a mixture, it is not possible to visually recognize which ion signal belongs to which protein, although this mixture contains only four proteins. Methods for charge deconvolution do, however, exist, for example the well-known program “MaxEnt” (maximum entropy charge deconvolution), which uses an entropy definition to compute the most probable deconvoluted mass spectrum for the measured mass spectrum. The result of such a deconvolution of the mass spectrum in FIG. 3 is shown in FIG. 4; the four proteins of the mixture are clearly recognizable. Unfortunately, the “MaxEnt” program requires one to two minutes for the deconvolution on fast and powerful computers, and therefore cannot be used for a fast real-time search for suitable candidates for the fragmentation, especially not when several substances of a mixture have approximately the same retention times and thus elute simultaneously and unresolved from the chromatograph.
For the fragmentation, the simplest method according to the Prior Art simply first fragments the ion species with the highest intensity and acquires its fragment ion mass spectrum, then the ion species with the second highest intensity and so on. This means, however, that frequently ions of the same proteins, but with different charge numbers, are measured again and again before a second protein is finally found which differs from the first. Proteins of lower intensity (such as the insulin ions in FIGS. 3 and 4) are often not found at all. A second method according to the Prior Art also selects the ion species in the order of the intensities, but analyzes the separations of the isotopic signals to determine the charge z of this ion species, and from this the mass M of the protein via the known M/z. If the second highest ion species has the same mass M, it is not selected for the fragmentation, but instead the third highest ion species is investigated, etc. However, this method can only be used when the individual isotopic signals are well separated from each other, i.e. when the mass resolution of the mass analyzer used is sufficiently high; it fails in mass spectrometers which do not have an extremely high resolving power. It is almost impossible to use the method for masses above 30 kilodaltons because they require resolving powers of more than R=60 000, and realizing this with time-of-flight mass spectrometers is a challenge.
There is therefore a need for methods which can rapidly select the most suitable ion species for fragmentation from a complex mass spectrum of mixed proteins. FIG. 3 shows an example of such a complex mass spectrum. The selection should either cover each biopolymer involved, or a pre-determined number of biopolymers. A complete deconvolution is not required. It is advantageous to also determine the width of the isotopic distribution Δ(M/z) in order to achieve the optimum setting for the mass filter for the isolation of this ion species, for example.